Free radicals are highly reactive molecules having unpaired electrons in their outer electronic orbit. For example, hydrogen peroxide (H2O2) is an oxidizing agent which in the presence of organic matter, or if permitted to become alkaline, decomposes to oxygen and water. The dissociation reaction of hydrogen peroxide in an aqueous solution is as follows: 2H2O2˜2H2O+O2.
O2 is a gas at standard temperature and pressure (STP). If this gas is present in solution, the gas has a valency of two. Accordingly, it has two spare electrons to form a bond with another element, such as oxygen, carbon or a metal. For example, molecular structure of O2 is O═O. However at any given time a very small proportion of the oxygen in solution will dissociate to form free oxygen atoms O. However, these free oxygen atoms or radicals now carry a-ve charge and are extremely unstable. In effect they must combine with another oxygen free radical to form an oxygen molecule, or they will react with organic matter by splitting carbon double bonds, or with metals to form a metal oxide.
Hydrogen peroxide is found in natural water, such as sea water, or rain water, where it is an important species in redox reactions, in industrial processes, and in biological tissues, including blood, as a result of enzymatic reactions. Direct detection of hydrogen peroxide is an important analytical task, and numerous techniques have been devised for measurement of hydrogen peroxide levels in fluids as indications of medical conditions, environmental quality, or the presence of pathogens in cells of both animals and plants.
Superoxide radicals (O2.−) in living tissue can be derived from many sources, such as activated granulocytes, endothelial cells, xanthine oxidase-catalyzed reactions, mitochondrial metabolism, and transition metal reactions with oxygen. Hydrogen peroxide (H2O2) can be produced from the dismutation of superoxide radicals (O2.) catalyzed by the enzyme superoxide dimutase (SOD), from transition metal reactions with superoxide radicals, and from enzymes (e.g., glycollate oxidase and urate oxidase) which produce peroxide directly without first producing superoxide. The presence of antioxidants, including certain enzymes such as SOD and catalase, serves to limit the concentration of the reactive oxygen species in plasma and tissues. Therefore, an increase in either the production of free radicals and/or a decrease in antioxidants can cause oxidative stress, contributing to possible cardiovascular complications in animals and humans. Similarly, oxygen free radicals may affect vascular resistance by inactivating nitric oxide (NO), thereby causing arteriolar vasoconstriction and elevation of peripheral hemodynamic resistance. Other conditions have also been associated with oxidative stress, including arthritis, acceleration of the progression of HIV to full-blown AIDS, and neurological diseases such as ALS.
Oxygen free radicals and related intermediates have also been suggested as playing a role in hypertension and may play a role by affecting vascular smooth muscle contraction and resistance to blood flow. In individuals with histories of conditions such as atherosclerosis, stroke and myocardial infarction, hypertension constitutes a risk factor.
A number of different techniques are known for measurement of oxygen free radicals and their intermediates. These methods include the use of electrodes, chemiluminescence, and fluorescence. These methods are all limited to measuring oxygen free radicals from stimulated neutrophils or deproteinized whole blood.
A hydrogen peroxide sensing system for measuring hydrogen peroxide in plasma is disclosed in published PCT patent application No. WO/1999/015891 entitled “SYSTEM AND METHOD FOR MEASURING HYDROGEN PEROXIDE LEVELS IN A FLUID AND METHOD FOR ASSESSING OXIDATIVE STRESS,” inventors Lacy et al. In the disclosed system the test sample of plasma from a fluid or fluid-containing material which is to be analyzed for hydrogen peroxide content is divided into two equal portions and a hydrogen peroxide oxidation sensor is inserted into each portion. An inhibitor for the enzyme catalase, such as sodium azide, is added to one of the portions to stabilize the hydrogen peroxide present. A quantity of catalase is added to the other portion to deplete any hydrogen peroxide present by catalyzing it to oxygen. Hydrogen peroxide oxidation of each portion at the respective sensor is then measured, along with background oxidation of any other oxidizable species in the sample. The signal from the sensor in the depleted hydrogen peroxide sample is subtracted from the signal from the stabilized hydrogen peroxide sample to eliminate the signals' contributions from background oxidation, thus yielding a resultant signal which is representative of the amount of hydrogen peroxide production in the subject fluid or material.
While the system disclosed in WO/1999/015891 may have limited utility in certain applications, it requires two separate portions of the plasma from the sample fluid or material as well as chemical treatment of each of the portions. Such a system is useful primarily in a laboratory where there are facilities for chemically treating the portions, and where supplies of the treating chemicals can be made available. This disclosed system is not particularly useful for analysis of samples in the field or where the treating chemicals are not conveniently available. This system also does not account for the fact that either or both of the treating agents may affect other components of the samples so that the two samples may end up being different from each other with respect to more than just the hydrogen peroxide component. Further, since the treating chemicals or enzymes must be added to each sample, the system must be recalibrated for each run.
U.S. Pat. No. 6,592,746 to Schmid-Schoenbein issued on Jul. 15, 2003 discloses a sensor probe for determining hydrogen peroxide concentration. The disclosed sensor probe can measure the hydrogen peroxide content of a single sample using two oxygen sensors whose electrodes are encased in defined membranes. The oxygen reference sensor is encased in a hydrophobic membrane which prevents the transport of hydrogen peroxide or electrochemical poisons or interferents and isolates the electrodes and an electrolyte fluid surrounding the electrodes from the sample fluid. The hydrogen-peroxide-generated oxygen (HPGO) sensor is also is encased in such a hydrophobic membrane, but has in series with and distally of the hydrophobic membrane a hydrophilic membrane which contains an immobilized enzyme such as catalase, peroxidase or other enzymes of a family which catalyzes the reaction of hydrogen peroxide to oxygen and water. At the HPGO sensor, the hydrogen peroxide is catalyzed to oxygen by the enzyme so that the HPGO sensor measures an enhanced concentration of oxygen relative to the oxygen reference sensor. The signals of each of the oxygen sensors are sent to a summer, which subtracts the equal background oxygen concentration of both, yielding a resultant difference signal representative of the concentration of hydrogen peroxide content of the sample itself. A suitable display or data collection device is used to capture the information in visible or digital form. Methods of use of the device for determining hydrogen peroxide content of human or animal tissues or fluids, or environmental or industrial fluids or fluid-containing materials, are also disclosed. Unfortunately, the immobilized enzyme-based probe disclosed by Schmid-Schoenbein have poor durability due to causes including the leaching of proteins during sensing. In addition, the enzyme-based probes require special care for storage, including generally low temperatures and wet conditions.